A new domestic cat genome assembly based on long sequence reads empowers feline genomic medicine and identifies a novel gene for dwarfism.

The domestic cat (Felis catus) numbers over 94 million in the USA alone, occupies households as a companion animal, and, like humans, suffers from cancer and common and rare diseases. However, genome-wide sequence variant information is limited for this species. To empower trait analyses, a new cat genome reference assembly was developed from PacBio long sequence reads that significantly improve sequence representation and assembly contiguity. The whole genome sequences of 54 domestic cats were aligned to the reference to identify single nucleotide variants (SNVs) and structural variants (SVs). Across all cats, 16 SNVs predicted to have deleterious impacts and in a singleton state were identified as high priority candidates for causative mutations. One candidate was a stop gain in the tumor suppressor FBXW7. The SNV is found in cats segregating for feline mediastinal lymphoma and is a candidate for inherited cancer susceptibility. SV analysis revealed a complex deletion coupled with a nearby potential duplication event that was shared privately across three unrelated cats with dwarfism and is found within a known dwarfism associated region on cat chromosome B1. This SV interrupted UDP-glucose 6-dehydrogenase (UGDH), a gene involved in the biosynthesis of glycosaminoglycans. Importantly, UGDH has not yet been associated with human dwarfism and should be screened in undiagnosed patients. The new high-quality cat genome reference and the compilation of sequence variation demonstrate the importance of these resources when searching for disease causative alleles in the domestic cat and for identification of feline biomedical models.

Hotspot Exon 15 Mutations in BRAF Are Uncommon in Feline Tumours.

BRAF is one of multiple RAF proteins responsible for the activation of the MAPK cell signalling cascade involved in cell growth, differentiation, and survival. A hotspot BRAFV600E mutation, in exon 15, was determined to be a driver in 100% hairy cell leukaemias, 50%-60% of human melanomas, 30%-50% of human thyroid carcinomas and 10%-20% of human colorectal carcinomas. The orthologous BRAFV595E mutation was seen in 67% and 80% of canine bladder transitional cell carcinomas and prostatic adenocarcinomas, respectively. Since veterinary and human cancers exploit similar pathways and BRAF is highly conserved across species, BRAF can be expected to be a driver in a feline cancer. Primers were developed to amplify exon 15 of feline BRAF. One hundred ninety-six feline tumours were analysed. Sanger sequencing of the 211 bp PCR amplicon was done. A BRAF mutation was found in one tumour, a cutaneous melanoma. The mutation was a BRAFV597E mutation, orthologous to the canine and human hotspot mutations. A common synonymous variant, BRAFT586T, was seen in 23% (47/196) of tumours. This variant was suspected to be a single nucleotide polymorphism. BRAF was not frequently mutated in common feline tumours or in tumour types that frequently harbour BRAF mutations in human and canine cancers. As is seen in canine cancer genomics, the mutational profile in feline tumours may not parallel the histologic equivalent in human oncology.

T1ρ, T2 mapping, and EPIC-µCT Imaging in a Canine Model of Knee Osteochondral Injury.

The dog is the most commonly used large animal model for the study of osteoarthritis. Optimizing methods for assessing cartilage health would prove useful in reducing the number of dogs needed for a valid study of osteoarthritis and cartilage repair. Twelve beagles had critical-sized osteochondral defects created in the medial femoral condyle of both knees. Eight dogs had T1ρ and T2 magnetic resonance imaging (MRI) performed approximately 6 months after defect creation. Following MRI evaluations, all 12 dogs were humanely euthanatized and cartilage samples were obtained from the medial and lateral femoral condyles, medial and lateral tibial plateaus, trochlear groove, and patella for proteoglycan and collagen quantification. Equilibrium partitioning of an ionic contrast (EPIC)-µCT was then performed followed by the histologic assessment of the knees. Correlations between T1ρ, T2, EPIC-µCT and proteoglycan, collagen, and histology scores were assessed using a multivariate analysis accounting for correlations from samples within the same knee and in the same dog. Pearson's correlation coefficients were calculated to assess the strength of significant relationships. Correlations between µCT values and biochemical or histologic assessment were weak to moderately strong (0.09-0.41; p < 0.0001-0.66). There was a weak correlation between the T2 values and cartilage proteoglycan (-0.32; p = 0.04). The correlation between T1ρ values and cartilage proteoglycan were moderately strong (-0.38; p < 0.05) while the strongest correlation was between the T1ρ values and histological assessment of cartilage with a correlation coefficient of 0.58 (p < 0.0001). These data suggest that T1ρ shows promise for possible utility in the translational study of cartilage health and warrants further development in this species. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:368-377, 2020.

Oral squamous cell carcinoma in a Vietnamese pot-bellied pig (Sus scrofa).

An 18-year-old, neutered, male Vietnamese pot-bellied pig (Sus scrofa) was treated for chronic, intermittent nasal discharge and sneezing. The animal was diagnosed with severe periodontal disease (grade IV), an oronasal fistula, and multiple tooth root abscesses via dental examination and computed tomography of the skull. Dentistry was performed, including multiple tooth extractions, and antibiotic therapy was initiated. Eighteen months later, the animal was evaluated for lethargy, anorexia, and a firm, 12 cm x 12 cm mass between the 2 rami of the mandible. Laboratory testing revealed moderate anemia, severe leukocytosis, and hyperglobulinemia. Skull radiographs indicated osteomyelitis of the mandible and soft-tissue swelling. A fine-needle aspirate and biopsy were taken, and results were consistent with squamous cell carcinoma. Treatment with piroxicam and antibiotics was initiated as needed to control signs of pain and secondary infection, respectively. Three months after diagnosis, the pig was euthanized due to cachexia and severe depression secondary to squamous cell carcinoma. On postmortem examination, the right mandibular area contained multiple, coalescing, irregular masses extending from the ramus rostrally to the mandibular canine teeth and ventrally within the intermandibular space, completely obliterating the normal anatomy. An open midshaft fracture was present on the right mandible. On histopathology, the masses were confirmed as locally invasive and destructive squamous cell carcinoma. No evidence of metastasis was noted in regional lymph nodes or in any of the distant sites evaluated.

Impact of Multilevel Facetectomy and Rod Curvature on Anatomical Spinal Reconstruction in Thoracic Adolescent Idiopathic Scoliosis.

STUDY DESIGN: A prospective, nonrandomized study. OBJECTIVE: The aim of this study was to assess surgical outcomes of multilevel facetectomy and rod curvature with simultaneous double-rod rotation technique for anatomical spinal reconstruction in thoracic adolescent idiopathic scoliosis (AIS). SUMMARY OF BACKGROUND DATA: Although some surgical techniques maintain or restore thoracic kyphosis (TK), next-generation strategies for thoracic AIS should include corrections in three anatomical planes. METHODS: The study included 39 consecutive patients with Lenke 1 or Lenke 2 thoracic AIS treated at our institution. After all-level facetectomy at instrumentation level, except for the lowest intervertebral segment, two rods were identically bent to guide postoperative anatomical TK without reference to the intraoperative coronal alignment of the AIS deformity. Outcome measures included patient demographics, radiographic measurements, and Scoliosis Research Society (SRS) questionnaire scores. RESULTS: After 2 years of follow-up, the average main thoracic Cobb angle correction rate was 83.5%, and the final correction loss was 2.2°. The average preoperative TK (T5-T12) significantly increased from 13.2° to 24.6° (P < 0.001) at final follow-up. The percentage of patients with a T6-T8 location of the TK apex significantly increased from 51.3% preoperatively to 87.2% at final follow-up. The average preoperative vertebral rotation angle significantly decreased from 18.7° to 12.8° postoperatively (P < 0.001). The average preoperative total SRS questionnaire score significantly increased from 3.5 to 4.5 (P < 0.001) at final follow-up. There was no implant breakage and vascular and neurologic complications, with all patients demonstrating solid fusion at final follow-up. CONCLUSION: Multilevel facetectomy and rod curvature play an important role in anatomical spinal reconstruction in patients with thoracic AIS. From the spatiotemporal point of view, four-dimensional correction could be actively performed by rod curvature under multilevel facetectomy and is expected to obtain an anatomical thoracic spine postoperatively, indicating that an anatomically designed rod could be supplied as a pre-bent rod. LEVEL OF EVIDENCE: 3.

Does Anterior Cruciate Ligament Innervation Matter for Joint Function and Development of Osteoarthritis?

Complications following anterior cruciate ligament (ACL) injury and reconstruction that include chronic dysfunction, second ACL injury, and posttraumatic osteoarthritis (OA) may be interrelated and stem from the inability to fully restore native ACL integrity and function. The loss of ACL sensory input following injury may significantly contribute to joint dysfunction. We developed a novel preclinical animal model to assess the contributions of ACL sensory afferents to knee joint function and health. We hypothesized that ACL sensory denervation would manifest in knee joint dysfunction and development of early OA. Purpose-bred, adult research dogs (n = 9 dogs, 18 knees) underwent arthroscopic surgery to create three treatment groups: (1) partial ACL tear, (2) ACL denervated, and (3) whole-joint denervated. A neurotoxin injected directly into the ACL or into the joint space was used to induce sensory denervation, and sham procedures were done on contralateral knees. After intervention, dogs participated in a regimented exercise program. Gait analysis and clinically relevant functional assessments were performed. At week 12, the animals were humanely euthanatized for arthroscopic, gross, and histologic assessments. ACL partial tear group demonstrated the greatest overall knee dysfunction. Clinical measures of function revealed a significant difference between the ACL partial tear and ACL denervated group (p < 0.05), but these differences were not observed between the ACL and whole-joint denervated groups (p > 0.05). A significant reduction in limb loading was experienced by the ACL partial tear group compared with controls (p < 0.05) but not between other groups. Arthroscopic evaluation found no evidence for overt articular cartilage damage, meniscal pathology, or osteophyte formation was noted in any group. No significant differences (p > 0.05) were observed in ACL pathology and OA severity scores between the ACL partial tear and the ACL denervated groups. The results of our study indicate that ACL sensory loss may contribute to joint dysfunction and subsequent OA changes. Further investigation and development of this model are important to improve clinical therapies and optimize patient outcomes following ACL injury.

In vivo bone tunnel evaluation of nanoparticle-grafts using an ACL reconstruction rabbit model.

Acellular human gracilis tendons conjugated with gold nanoparticles (AuNP) and hydroxyapatite nanoparticles (nano-HAp) were used as a graft in an anterior cruciate ligament (ACL) reconstruction rabbit model. The ACLs of 11 New Zealand rabbits were reconstructed using grafts conjugated without nanoparticles, with AuNP only, and with both AuNP and nano-HAp. Semi-quantitative histological scoring of bone tunnel portion of grafts was performed after 14 weeks. Bone tunnels were scored for graft degeneration, graft remodeling, percentage of new host fibrous connective, collateral connection, head-to-head connection, graft collagen fiber organization, new host fibrous connective tissue organization, and graft and interface vascularity. All grafts were intact at 14 weeks. Results of bone tunnel scoring indicate remodeling in all graft types with new organized host fibrous connective tissue, head-to-head connection to bone and mild inflammation associated with remodeling. Components of the 20 nm AuNP grafts have significantly more graft degeneration, more new host fibrous connective tissue, and more vascularity compared to crosslinked grafts. Comparison between femoral and tibial tunnel scores indicate more degeneration in femoral tunnels compared to tibial tunnels. Overall results indicated potentially enhanced remodeling from the use of 20 nm AuNP grafts. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1071-1082, 2017.

Surgical treatment of Lenke 1 thoracic adolescent idiopathic scoliosis with maintenance of kyphosis using the simultaneous double-rod rotation technique.

STUDY DESIGN: Retrospective analysis of a prospectively collected, consecutive, nonrandomized series of patients. OBJECTIVE: To assess the surgical outcomes of the simultaneous double-rod rotation technique for treating Lenke 1 thoracic adolescent idiopathic scoliosis (AIS). SUMMARY OF BACKGROUND DATA: With the increasing popularity of segmental pedicle screw spinal reconstruction for treating AIS, concerns regarding the limited ability to correct hypokyphosis have also increased. METHODS: A consecutive series of 32 patients with Lenke 1 main thoracic AIS treated with the simultaneous double-rod rotation technique at our institution was included. Outcome measures included patient demographics, radiographical measurements, and Scoliosis Research Society questionnaire scores. RESULTS: All 32 patients were followed up for a minimum of 2 years (average, 3.6 yr). The average main thoracic Cobb angle correction rate and the correction loss at the final follow-up were 67.8% and 3.3°, respectively. The average preoperative thoracic kyphosis (T5-T12) was 11.9°, which improved significantly to 20.5° (P < 0.0001) at the final follow-up. An increase in thoracic kyphosis was significantly correlated with an increase in lumbar lordosis at the final follow-up (r = 0.42). The average preoperative vertebral rotation angle was 19.7°, which improved significantly after surgery to 14.9° (P = 0.0001). There was no correlation between change in thoracic kyphosis and change in apical vertebral rotation (r =-0.123). The average preoperative total Scoliosis Research Society questionnaire score was 3.0, which significantly improved to 4.4 (P < 0.0001) at the final follow-up. Throughout surgery and even after, there were no instrumentation failures, pseudarthrosis, infection of the surgical site, or clinically relevant neurovascular complications. CONCLUSION: The simultaneous double-rod rotation technique for treating Lenke 1 AIS provides significant sagittal correction of the main thoracic curve while maintaining sagittal profiles and correcting coronal and axial deformities. LEVEL OF EVIDENCE: 4.